目的探索载脂蛋白E模拟肽COG1410对小鼠视网膜缺血－再灌注(IR)损伤后M1/M2小胶质细胞极化及视网膜神经节细胞(RGCs)存活的影响及其可能的机制。
方法将18只8周龄C57BL/6J雄性小鼠按照随机数字表法分为正常对照组6只、IR 3 d组6只、IR 7 d组3只和IR 14 d组3只，其中IR组小鼠使用生理盐水进行前房灌注，将眼压提高至100 mmHg(1 mmHg＝0.133 kPa)并维持1 h，以建立视网膜IR损伤模型。取正常对照组和IR 3 d组各3只，采用视网膜冰冻切片免疫荧光染色法观察视网膜小胶质细胞的分布情况。取正常对照组、IR 3 d组、IR 7 d组和IR 14 d组各3只，通过视网膜铺片免疫荧光染色法观察视网膜M1型、M2型小胶质细胞随IR损伤后时间的变化。另取91只C57BL/6J小鼠按照随机数字表法随机分为正常对照组19只、IR组24只、生理盐水组24只、COG1410组24只，其中正常对照组小鼠维持正常眼压，其余3个组均建立IR损伤模型，且COG1410组和生理盐水组在造模后分别尾静脉注射1 mg/kg COG1410和等体积生理盐水。采用视网膜铺片免疫荧光染色法观察各组小胶质细胞表型及RGCs存活率；采用实时荧光定量PCR法检测各组视网膜肿瘤坏死因子α(TNF-α)、白细胞介素-1β(IL-1β)mRNA的相对表达量；采用TUNEL法观察视网膜神经细胞的凋亡情况；采用Western blot法检测各组视网膜核因子κB(NF-κB)、B淋巴细胞瘤2(Bcl-2)、Bcl-2关联X蛋白(Bax)蛋白的表达水平。
结果正常对照组与IR 3 d组视网膜小胶质细胞主要分布在神经节细胞层、内丛状层和外丛状层。正常对照组、IR 3 d组、IR 7 d组和IR 14 d组M1型、M2型小胶质细胞数目总体比较差异均有统计学意义( F＝29.83、57.62；均 P<0.001)；与正常对照组相比，IR 3 d组M1型小胶质细胞数量增多，IR 7 d组M2型小胶质细胞数量增多，差异均有统计学意义(均 P<0.05)。正常对照组、IR组、生理盐水组、COG1410组M1型小胶质细胞比例分别为(4.25±0.57)%、(65.26±10.43)%、(63.01±4.93)%和(33.13±4.46)%，M2型小胶质细胞的数目分别为(4.50±0.20)%、(11.47±0.24)%、(11.75±0.17)%和(38.93±4.26)%，总体比较差异均有统计学意义( F＝23.33、50.82；均 P<0.05)；与IR组比较，COG1410组M1型小胶质细胞数目减少而M2型小胶质细胞数目增多，差异均有统计学意义(均 P<0.05)；各组RGCs存活率及视网膜TNF-α mRNA、IL-1β mRNA相对表达量、凋亡细胞数目、NF-κB和Bax蛋白表达水平、Bax/Bcl-2值总体比较差异均有统计学意义( F＝30.77、12.52、6.74、28.72、13.02、7.94、7.58，均 P<0.05)；与正常对照组比较，IR组RGCs存活率明显降低，视网膜凋亡细胞数增多，TNF-α和IL-1β mRNA表达量升高，视网膜NF-κB和Bax蛋白表达水平升高，Bax/Bcl-2比值升高，差异均有统计学意义(均 P<0.05)；与IR组比较，COG1410组视网膜RGCs存活率升高，TNF-α和IL-1β mRNA表达量降低，TUNEL阳性细胞减少，NF-κB和Bax蛋白表达水平降低、Bax/Bcl-2比值降低，差异均有统计学意义(均 P<0.05)。
结论视网膜IR后3 d，COG1410促进M1型小胶质细胞向M2型极化，抑制视网膜NF-κB及下游炎症因子表达，减轻视网膜炎症反应，同时抑制凋亡相关蛋白的表达，从而促进RGCs存活。
ObjectiveTo explore the effects of apolipoprotein E-mimetic peptide COG1410 on M1/M2 microglia polarization and retinal ganglion cells (RGCs) survival after ischemia-reperfusion (IR) injury in the mouse retina and its possible mechanisms.
MethodsEighteen 8-week-old C57BL/6J male mice were divided into control group (6 mice), IR 3 days group (6 mice), IR 7 days group (3 mice), and IR 14 days group (3 mice) according to the randomized number table method.Mice in IR group were perfused in the anterior chamber using saline, and the intraocular pressure (IOP) was raised to 100 mmHg (1 mmHg=0.133 kPa) and maintained for 1 hour in order to establish a model of IR injury in the retina.Three mice from control group and 3 mice from IR 3 days group were taken to observe the distribution of retinal microglia by immunofluorescence staining of retinal frozen sections.Three mice were taken from normal control, IR 3 days, IR 7 days, and IR 14 days groups respectively to observe the changes of retinal M1-type and M2-type microglial cells with time after IR injury by immunofluorescence staining of retina.Another 91 C57BL/6J mice were randomly divided into normal control group (19 mice), IR group (24 mice), saline group (24 mice), and COG1410 group (24 mice) according to the random number table method.Mice in normal control group maintained a normal IOP, and the IR injury model was established in the other three groups.In addition, COG1410 group and saline group were injected with 1 mg/kg COG1410 and an equal volume of saline by tail vein injection, respectively.The microglia phenotype and survival rate of RGCs were observed by immunofluorescence staining of retinal wholemount.The relative expressions of retinal tumor necrosis factor-ɑ (TNF-ɑ) and interleukin-1β (IL-1β) mRNA were detected by real-time fluorescence quantitative PCR.The apoptosis of retinal neuronal cells was observed by the TUNEL assay.The expression levels of retinal nuclear factor-κB (NF-κB), B lymphocyte-2 (Bcl-2), and Bcl-2-associated X protein (Bax) proteins were detected by Western blot.Use and care of animals strictly complied with the Hubei Provincial Regulations on the Management of Laboratory Animals and the experiment was approved by the Animal Ethics Committee of the Renmin Hospital of Wuhan University (No.WDRM20190113).
ResultsRetinal microglia in normal control group and IR 3 days group were mainly distributed in the ganglion cell layer, inner plexiform layer, and outer plexiform layer.There were statistically significant differences in the comparison of the proportions of M1-type and M2-type microglia among normal control, IR 3 days, IR 7 days, and IR 14 days groups ( F=29.83, 57.62; both at P<0.001). Compared with normal control group, the number of M1-type microglia was higher in IR 3 days group, and the number of M2-type microglia was higher in IR 7 days group, and the differences were statistically significant (all at P<0.05). The proportions of M1-type microglia in normal control group, IR group, saline group, and COG1410 group were (4.25±0.57)%, (65.26±10.43)%, (63.01±4.93)%, and (33.13±4.46%), respectively, and the proportions of M2-type microglia in the four groups were (4.50±0.20)%, (11.47±0.24 )%, (11.75±0.17)%, and (38.93±4.26)%, showing statistically significant differences among them ( F=23.33, 50.82; both at P<0.001). The proportions of M1-type microglia decreased while the proportions of M2-type microglia increased in COG1410 group when compared with IR group, and the differences were statistically significant (both at P<0.05). There were statistically significant differences in RGCs survival rate, relative expression of retinal TNF-ɑ and IL-1β mRNA, retinal apoptotic cell count, retinal NF-κB and Bax protein expression levels, and Bax/Bcl-2 ratio among the four groups ( F=30.77, 12.52, 6.74, 28.72, 13.02, 7.94, 7.58; all at P<0.05). Compared with normal control group, there were significant decreases in the survival rate of RGCs and increases in retinal apoptotic cell number, TNF-ɑ and IL-1β mRNA expression, retinal NF-κB and Bax protein expression levels, and Bax/Bcl-2 ratio in IR group (all at P<0.05). Compared with IR group, the COG1410 group had increased retinal RGCs survival rate, decreased TNF-ɑ and IL-1β mRNA expression levels, decreased TUNEL-positive cells, decreased NF-κB and Bax proteins expression levels, and decreased Bax/Bcl2 ratio, and the differences were statistically significant (all at P<0.05).
ConclusionsThree days after retinal IR modeling, COG1410 promotes the polarization of M1-type microglia to M2-type, inhibits the expression of retinal NF-κB and downstream inflammatory factors, and attenuates the retinal inflammatory response, as well as inhibits the expression of apoptosis-related proteins, which promotes the survival of RGCs.
赵茹,罗晋媛,贺涛,等. COG1410对视网膜缺血－再灌注损伤小鼠视网膜神经节细胞的保护作用及其机制[J]. 中华实验眼科杂志,2023,41(11)：1065-1075.
DOI:10.3760/cma.j.cn115989-20221116-00534版权归中华医学会所有。
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赵茹：酝酿和设计实验、实施研究、采集数据、分析/解释数据、文章撰写；罗晋媛：实施研究、采集数据、分析/解释数据；贺涛：参与实验设计、论文指导；邢怡桥：实验设计、论文审阅及定稿
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